High-Throughput Sequencing Reveals the Loss-of-Function Mutations in GALT Cause Recessive Classical Galactosemia

Evaluation and rational design of guide RNAs for efficient CRISPR/Cas9-mediated mutagenesis in Ciona

10.1101/041632 ◽

2016 ◽

Cited By ~ 3

Author(s):

Shashank Gandhi ◽

Maximilian Haeussler ◽

Florian Razy-Krajka ◽

Lionel Christiaen ◽

Alberto Stolfi

Keyword(s):

High Throughput ◽

Rational Design ◽

Genome Engineering ◽

High Throughput Sequencing ◽

Loss Of Function ◽

Tissue Specific ◽

Guide Rna ◽

Guide Rnas ◽

Highly Active

AbstractThe CRISPR/Cas9 system has emerged as an important tool for various genome engineering applications. A current obstacle to high throughput applications of CRISPR/Cas9 is the imprecise prediction of highly active single guide. RNAs (sgRNAs). We previously implemented the CRISPR/Cas9 system to induce tissue-specific mutations in the tunicate Ciona. In the present study, we designed and tested 83 single guide RNA (sgRNA) vectors targeting 23 genes expressed in the cardiopharyngeal progenitors and surrounding tissues of Ciona embryo. Using high-throughput sequencing of mutagenized alleles, we identified guide sequences that correlate with sgRNA mutagenesis activity and used this information for the rational design of all possible sgRNAs targeting the Ciona transcriptome. We also describe a one-step cloning-free protocol for the assembly of sgRNA expression cassettes. These cassettes can be directly electroporated as unpurified PCR products into Ciona embryos for sgRNA expression in vivo, resulting in high frequency of CRISPR/Cas9-mediated mutagenesis in somatic cells of electroporated embryos.We found a strong correlation between the frequency of an Ebf loss-of-function phenotype and the mutagenesis efficacies of individual Ebf-targeting sgRNAs tested using this method. We anticipate that our approach can be scaled up to systematically design and deliver highly efficient sgRNAs for the tissue-specific investigation of gene functions in Ciona.

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Deep Mutational Scan of a cardiac sodium channel voltage sensor

10.1101/773887 ◽

2019 ◽

Cited By ~ 1

Author(s):

Andrew M. Glazer ◽

Brett M. Kroncke ◽

Kenneth A. Matreyek ◽

Tao Yang ◽

Yuko Wada ◽

...

Keyword(s):

Ion Channel ◽

Sodium Channel ◽

High Throughput ◽

High Throughput Sequencing ◽

Voltage Sensor ◽

Patch Clamping ◽

Loss Of Function ◽

Pathogenic Variants ◽

Cardiac Sodium Channel

AbstractVariants in ion channel genes have classically been studied in low-throughput by patch clamping. Deep Mutational Scanning (DMS) is a complementary approach that can simultaneously assess function of thousands of variants. We have developed and validated a method to perform a DMS of variants in SCN5A, which encodes the major voltage-gated sodium channel in the heart. We created a library of nearly all possible variants in a 36 base region of SCN5A in the S4 voltage sensor of domain IV and stably integrated the library into HEK293T cells. In preliminary experiments, challenge with three drugs (veratridine, brevetoxin, and ouabain) could discriminate wildtype channels from gain and loss of function pathogenic variants. High-throughput sequencing of the pre- and post-drug challenge pools was used to count the prevalence of each variant and identify variants with abnormal function. The DMS scores identified 40 putative gain of function and 33 putative loss of function variants. For 8/9 variants, patch clamping data was consistent with the scores. These experiments demonstrate the accuracy of a high-throughput in vitro scan of SCN5A variant function, which can be used to identify deleterious variants in SCN5A and other ion channel genes.

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